The objective of the proposed study is to determine the mechanism of killing of cells exposed to long duration, moderate hyperthermia (LDMH) (e.g. 41.5 degrees C - 42.5 degrees C for 48-72 hrs). One possible obstacle to achieving significant cell killing during LDMH is the expression of chronic thermotolerance, defined as resistance to further heat-induced cell killing which appears during the heating interval. Our preliminary results indicate that under some conditions chronic thermotolerance is not expressed at 41.5 degrees C in human cells. Accordingly, we will determine if LDMH can be an effective method of cell killing by examining the response of a series of human normal and tumor cell lines which possess varying degrees of chronic thermotolerance expression and tumorigenicity (Specific Aim 1). Previous results indicate that cell death during LDMH is associated with an accumulation of cells in S phase and the appearance of premature mitotic events. The onset of mitosis is known to be regulated by a biochemical oscillator. Therefore, we will determine if these premature mitotic events occurring during LDMH are due to an advancement of the timing of this oscillator, or if LDMH delays the completion of S, with entry into mitosis occurring on schedule (Specific Aim 2). Finally, we will determine if agents which delay the onset of either S phase or mitosis act to protect cells from LDMH-induced cell killing (Specific Aim 3). one advantage of the use of LDMH in cancer therapy lies in the fact that the desired temperature range is clinically-achievable. Knowledge of the cellular mechanism of chronic thermotolerance expression, combined with readily measurable indicators of LDMH-induced cell killing (i.e., entry into S or premature mitotic events), should be of benefit in its clinical administration.